茶多酚对H_2O_2诱导大鼠晶状体上皮细胞中Caveolin-1表达的影响

Role of tea polyphenols in regulation of expression of Caveolin-1 in lens epithelial cells in rats treated with H_2O_2

目的建立离体过氧化氢(H2O2)诱导的大鼠白内障模型并观察茶多酚(tea polyphenols,TP)对H2O2诱导大鼠晶状体上皮细胞(lens epithelial cell,LEC)中小窝蛋白-1(Caveolin-1)表达的影响。方法采用离体晶状体培养技术,在一定浓度H2O2的MEM培养液中置入透明的SD大鼠晶状体,建立实验性白内障晶状体模型。设置空白对照组、H2O2组和TP(0.02mg·mL-1、0.20mg·mL-1、2.00mg·mL-1)处理组,分别于不同时间点(0h、6h、12h、24h、48h)取样,应用RT-PCR检测LEC中Caveolin-1 mRNA的表达,Western blot检测LEC中Caveolin-1蛋白的表达。结果 H2O2下调LEC中Caveolin-1表达,H2O2组加入H2O20h、6h、12h、24h、48hCaveolin-1 mRNA表达分别为0.740±0.210、0.560±0.130、0.350±0.160、0.204±0.142、0.197±0.133,呈时间依赖性,在H2O2作用24h时,Caveolin-1表达下调最明显(P<0.01),遂选定24h为后续实验时间点。不同浓度的TP均可抑制LEC中Caveolin-1表达的减少,24h时RT-PCR检测空白对照组、H2O2组、TP处理组(0.02mg·mL-1、0.20mg·mL-1、2.00mg·mL-1)Caveolin-1 mRNA表达分别为0.687±0.141、0.112±0.124、0.341±0.115、0.562±0.102、0.584±0.098,Western-blot检测空白对照组、H2O2组、TP处理组(0.02mg·mL-1、0.20mg·mL-1、2.00mg·mL-1)Caveolin-1蛋白表达分别为0.819±0.124、0.166±0.132、0.214±0.154、0.565±0.089、0.621±0.103,TP浓度为0.20mg·mL-1时抑制效果最显著(P<0.05)。结论 TP可抑制H2O2诱导的LEC中Caveolin-1的表达减少,且在TP浓度为0.20mg·mL-1时抑制效果最显著。

Objective To establish the model of rat cataract in vitro through induction of hydrogen peroxide （ H2 O2 ） and investigate the influence of tea polyphenols （TP） on the expression of Caveolin-1 in H2O2-induced lens epithelial cells （LEC）. Methods In vitro culture technology of lens was applied. Transparent lens of SD rats were dipped into H2O2 MEM solution of a certain concentration to establish the experimental model of rat cataract in lens. Five groups were set： blank control group, H2O2 group, and three TP groups （0.02 mg ·mL-1, 0.20 mg ·mL-1, 2.00 mg·mL-1 ）. Samplings were taken at five time-in-points：0 hour,6 hours,12 hours,24 hours and 48 hours after the establishment. Caveolin-1 mRNA expression and Caveolin-1 protein expression in LEC were detected by RT-PCR and Western-blot, respectively. Results H2O2 lowered the expression level of Caveolin-1. Expression levels of Caveolin-1 mRNA of H2O2 group at all the five time-in-points were 0.740 ±0.210,0. 560 ±0. 130,0. 350 ±0. 160, 0. 204 ±0. 142 and 0. 197 ± 0. 133, which were time-dependent. At 24 hours, the expression level of Caveolin-1 was the lowest （P 〈 0.01 ）,thus this time-in-point was chosen as the beginning of the subsequent experiment. TP of all concentrations inhibited the lowering of the expression level of Caveolin-1. RT-PCR results showed that expression levels of Caveolin-1 mRNA of all the five groups were 0. 687 ± 0. 141,0. 112 ± 0. 124, 0. 341 ± 0. 115,0. 562 ± 0. 102,0. 584 ±0. 098, respectively. Western-blot results showed that expression levels of Caveolin-1 protein of all the five groups were 0. 819 ± 0. 124,0. 166 ±0. 132,0.214 ±0. 154,0.565 _±0.089,0.62l ±0. 103 ,respectively. The inhibitory effect was most significant when TP was of the concentration 0.20 mg ·mL-1 （P 〈 0.05 ）. Conclusion TP can inhibit the lowering of the expression level of Caveolin-1 and its inhibitory effect is most significant when TP is of the concentration 0.20 mg ·mL-1.